Pulmonary emphysema, the major component leading to morbidity and mortality in chronic obstructive pulmonary disease (COPD), is the fourth leading cause of death in the United States. Approximately 15 million Americans are affected by COPD with an increasing incidence in women. Smoking is the major risk factor for COPD and accounts for over 90% of cases seen worldwide. Despite this finding, the standard treatment for COPD has changed little over the past 20-30 years.
The etiology of emphysema is multiplex with a variety of different injuries leading to the disease, including structural damage to the lung, defective proteinase inhibitors and diseases which result in overproduction of neutrophils within the lung (Aaron, 1983). Currently the major hypothesis for the pathogenesis of emphysema is the protease-antiprotease theory (Janoff, 1985). This states that an imbalance between the levels of degradative enzymes and their respective inhibitors damages the connective tissue matrix components of the lung.
Emphysema is estimated to affect 1.6 million Americans and has been shown to be directly related to cigarette smoking. Although the primary mechanism leading to lung destruction in this disease is believed to be secondary to an imbalance between proteases and antiproteases, direct evidence of an increase in destructive enzymes within the lung parenchyma has never been demonstrated. The hypothesis was based mainly on the induction of emphysema in animals through intratracheal instillation of nonspecific proteolytic enzymes (Gross et al., 1965). However, the major-focus of many studies has concentrated on elastase as the primary destructive protease due to two particular observations. First, the association between the absence of alpha-1 antitrypsin (an inhibitor of elastase) and emphysema was made through the characterization of the rare hereditary disease, alpha-1 antitrypsin deficiency (Laurell and Erikson, 1963). Secondly, pancreatic and leukocyte elastase produce an emphysema-like phenotype experimentally when instilled intratracheally into certain animal species (Janoff et al., 1977). By contrast, when bacterial collagenase was injected into the lungs of hamsters, no emphysematous lesions developed (Johansen and Pierce, 1972).
The degradation of elastin readily explains the early development of the disease in alpha-1 antitrypsin deficiency (Laurell and Erikson, 1963). However, this represents only 1% of all human patients who suffer from emphysema and the relationship of alpha-1 antitrypsin to the major form of human emphysema which is induced by cigarette smoking remains unclear. In fact, studies over the past 20 years have failed to demonstrate elastase excess or inhibitor deficiency in the development of other forms of human emphysema (for reviews, see Hance and Crystal, 1975). Even in alpha-1 antitrypsin deficiency human patients, there is no direct biochemical evidence for actual proteolysis of elastin in vivo (Hance and Crystal, 1975).
Most importantly perhaps, there are a number of discrepancies between the emphysema observed in the elastase-induced animal model and human emphysema. First, there are differences in the morphological phenotype which have been identified at the ultrastructural level. Secondly, the elastase-induced animals suffer from an acute severe hemorrhagic and inflammatory response which could induce a variety of nonspecific initiators of the emphysematous process (Hoidal et al., 1989). Thirdly, this acute form of emphysema induced after a single intratracheal instillation of enzyme is direct contrast to human emphysema which develops only after chronic insult to the tissue over a period of years. Overall, the lack of direct evidence has led a number of investigators to question the exact role of elastase in human emphysema (Hance and Crystal, 1975; Pickrell and Mauderly, 1981).
A major hypothesis for the pathogenesis of pulmonary emphysema is that alveolar destruction results from an imbalance between proteolytic enzymes and inhibitors in the lung parenchyma. However, direct evidence of an increase in destructive enzymes within the lung parenchyma is lacking.
Chronic obstructive pulmonary disease (COPD), consisting of emphysema and chronic bronchitis, is the fourth leading cause of death in the United States. Approximately 15 million Americans are affected by COPD and there is an increasing incidence in women. Smoking is the major risk factor for COPD and accounts for over 90% of cases seen worldwide. Despite this finding, there are no specific therapies available to limit or prevent these slowly progressive destructive changes (Snider).
Studies over the past 20 years have failed to demonstrate elastase excess or inhibitor deficiency in the development of human emphysema (for reviews, see Hance and Crystal, 1975,). Investigators have identified a variety of protease activity in the bronchoalveolar lavage of smokers. However, cigarette smoking is associated with a 5-10 fold increase in cells recovered by lavage and may not represent what is actually occurring within the lung parenchyma. The chronicity of emphysema and absence of smoking late in the disease make it difficult to implicate these enzymes in the progression of the disease process. This lack of direct evidence has led a number of investigators to question the exact role of elastase in human emphysema (Hance and Crystal, 1975; Pickrell and Mauderly, 1981).
We have previously demonstrated that chronic expression of human collagenase in transgenic mice causes emphysema (D""Armiento et al., 1992). The enzyme used in the animal studies is identical, immunologically and functionally, to the human collagenase of fibroblasts and macrohpages. Most significantly, both of these cell types have been shown to be present within the normal alveolar structure. The development of emphysema upon degradation of collagen in animals implicates this enzyme in the human disease. Despite the lack of evidence demonstrating elastase activity in patients with emphysema and the establishment of emphysema in transgenic mice with the over expression of collagenase (MMP-1), investigators have continued to focus on the role of elastolytic enzymes in the human disease (Shapiro, The pathogenesis of emphysema 30 years later). Most recently, researchers have identified a protease (metalloelastase) secreted by macrophages which is capable of degrading elastin and have hypothesized that this enzyme is responsible for human emphysema. The present study was therefore performed to determine if there was increased expression of collagenase or elastase in the lungs of human patients with emphysema as compared to normal controls.
Matrix metalloproteinase inhibitors are known to block the action of a family of enzymes, the matrix metalloproteinases (Brown, P. Advan. Enzyme Requl., Vol 35, pp 293-300 1995). In the earliest experiments in animal cancer models, matrix metalloproteinase inhibitors were shown to block the spread of cancer and as a result were regarded initially as anti-metastic agents. Further, a series of experiments have shown that, in addition to blocking cancer spread, these inhibitors can also block cancer growth. Experiments have demonstrated this activity for a synthetic matrix metalloproteinase inhibitor, batimastat BB94 (xe2x80x9cBatimastatxe2x80x9d)
The batimastat-collagenase complex is described in detail and the activities of batimastat analogues are discussed in the light of the protein-inhibitor interactions in Grams, F. Biochemistry 1995, 34. 14012-14020.
Synthesis of Batimastat (BB94) is described in Campion et al. (1990).
Other known metalloproteinase inhibitors include tetracycline and its derivatives including minocycline, and dilantin.
The subject invention provides a use of a metalloproteinase inhibitor for the preparation of a pharmaceutical composition for treating human pulmonary emphysema which comprises admixing the metalloproteinase inhibitor in an amount effective to treat human pulmonary emphysema and a pharmaceutically acceptable carrier.
The subject invention also provides A pharmaceutical composition which comprises a metalloproteinase inhibitor in an amount effective to to treat human pulmonary emphysema and a pharmaceutically acceptable carrier.
The subject invention provides a method for treating human pulmonary emphysema comprising administering a metalloproteinase inhibitor. The subject invention also provides a method for preventing human pulmonary emphysema comprising administering a metalloproteinase inhibitor.
The subject invention further provides the above-described methods wherein the metalloproteinase inhibitor is active against collagenase
The subject invention also provides a method of treating mammals afflicted with a condition associated with the presence of collagenase in the lung tissue, comprising the step of administering to the individual a therapeutically effective amount of a metalloproteinase inhibitor of the present invention in a pharmaceutically acceptable composition.
The subject invention also provides a method of treating a subject in need of such treatment comprising administering by inhalation a metalloproteinase inhibitor active against collagenase in an amount effective to treat emphysema.
We previously demonstrated that chronic expression of human collagenase in the parenchyma of transgenic mice causes emphysema (D""Armiento et al., 1992). The pathology seen in the transgenic mouse lungs is strikingly similar to the morphological changes observed in human emphysema. The enzyme expressed in the animal studies is identical, immunologically and functionally, to the human collagenase of fibroblasts and macrophages. Both of these cell types are present within the normal alveolar structure. These findings led us to hypothesize that collagenase overexpression in the parenchyma of the lungs of patients with emphysema may contribute to the development and progression of the disease. To test this hypothesis, the present study was performed to determine the level of expression of collagenase and elastase in the lungs of patients with emphysema as compared to controls.
Recently, a transgenic mouse was generated which overexpressed the human enzyme collagenase (MMP-1) in the lung and the animals developed emphysema. We therefore hypothesized that an increase in this enzyme in human lungs may lead to emphysema.
Although emphysema is believed to be due to an imbalance in protease activity within the lung, a molecular analysis of the lung paranchyma from patients with emphysema has never been undertaken. This is the first direct demonstration at the RNA and protein level of a degradative enzyme in human emphysema tissue. This study shows that collagenase expression is present in patients with human emphysema secondary to cigarette smoking and not in normal patients or in alpha-1 antitrypsin deficiency patients who have not smoked. In addition, this study demonstrates that elastase expression and activity is not present in the lungs of patients with smoking induced emphysema.
Emphysema tissue obtained from lung reduction surgery was examined for the presence of collagenase (MMP-1), gelatinase (MMP-9), neutrophil elastase and metalloelastase mRNA through reverse transcriptase-PCR (RT-PCR) and ribonuclease protection assays and compared with normal lung tissue. Samples were obtained from 23 emphysema patients, 3 patients with alpha 1 antitrypsin deficiency and 8 control patients ranging from 39-60, 39-46 and 19-55 years in age, respectively. The samples were also examined for the presence of collagenase and elastase enzyme activity.
Collagenase mRNA was identified in 22 of the 23 emphysema patients, 1 out of 3 alpha-1 antitrypsin patients and 0 out of 8 control patients samples. Six out of seven samples tested from emphysema patients demonstrated collagenase protein by ELISA analysis with none of the controls demonstrating protein or enzyme activity. Elastase activity was found in only one samples which was from a patient with alpha 1 antitrypsin deficiency.
Although investigators have assayed the BAL fluid in emphysema patients for the presence of degradative enzymes, the lung paranchyma has never been examined. This is the first direct demonstration at the RNA and protein level of a degradative enzyme in smoking induced human emphysema tissue. The present data strongly suggest that collagenase-1 and not elastase, is a critical enzyme involved in the pathogenesis of human emphysema. The observation of increased collagenase (MMP-1) expression in the lung presents for the first time the possibility that inhibition of this enzyme would be feasible in the treatment and prevention of this human lung disease. The observation of increased collagenase (MMP-1) expression in the lung of patients with emphysema presents the possibility that this enzyme could be a target in the treatment and prevention of disease.
This study reports the first demonstration, at the RNA and protein level, of a degradative enzyme in human emphysema tissue. These data demonstrate that collagenase (MMP-1) is expressed in patients with emphysema secondary to cigarette smoking and is absent in normal patients. In contrast, neutrophil elastase expression and activity could not be detected in the lungs of patients with smoking induced emphysema and normal controls.
It is well established that only about 15% of long-term smokers will develop emphysema, whereas many patients with emphysema will experience progressive airflow obstruction even after they have stopped smoking. Animal studies have demonstrated that exposure to cigarette smoke increases collagenase expression and collagen degradation in the lung (Pardo, Wright). Immunohistochemical studies demonstrate that the collagenase expression is present in the macrophages, alveolar lining cells and interstitium. Therefore, it is conceivable that in humans the lung parenchyma is modified during the time period of smoking such that the collagenase enzyme is expressed. The present study demonstrate that even after smoking has ceased in the emphysema patients the collagenase continues to be expressed. The reasons for continued collagenase expression after smoking cessation are presently unknown. Our findings suggest that this collagenase expression plays a role in the continued progression of emphysema after smoking cessation. In addition, it is therefore possible that the fibroblasts in certain patients who are predisposed to emphysema have a greater potential to secrete the collagenase when exposed to cigarette smoke.
The present data strongly suggest that collagenase-1 is a critical enzyme involved in the pathogenesis of smoking induced human emphysema even after smoking has ceased. The combination of this study and the demonstration of lung destruction in transgenic animals through the overexpression of collagenase (MMP-1) leads one to hypothesize that the inhibition of this enzyme may be a suitable target to slow or arrest the progression of emphysema. Controlled trials of metalloproteinase inhibitors in emphysema can further support this hypothesis and provide a novel therapeutic intervention in the treatment of emphysema.